The present disclosure relates to articles of manufacture which include, as at least one component thereof, microporous membrane operatively associated with highly electropositive solid phase hydrophilic materials useful for highly efficient and irreversible binding of nucleic acids, methods of fabricating such articles of manufacture, including microporous membrane, and methods of using such articles of manufacture including microporous membrane to amplify nucleic acids and to store the membrane having the bound nucleic acid for archival purposes.
Numerous techniques are known in the art for separating nucleic acids from liquid biological samples and amplifying the same. The vast majority of these techniques, however, are time consuming and plagued by complication, as described below.
Both physical and chemical methods are known for extracting nucleic acids from biological samples. For example, nucleic acids may be separated from other cellular debris by ultra-centrifugation using sucrose or cesium chloride density gradients, such separation being in accord with buoyant density or sedimentation coefficient. Chemical methods of separating nucleic acids include phenol extraction, ethanol precipitation, and chaotropic reagent extraction. Affinity columns incorporating agents such as ethidium bromide and ethidium-acrylamide have also been used to recover nucleic acids from free solution. Physiochemical methods for extracting nucleic acids are also known, such as by agarose or polyacrylamide gel electrophoresis wherein the negatively charged nucleic acid molecules move toward the anode with the larger molecules moving more slowly.
In many applications it is often necessary to amplify and/or detect certain nucleic acids of interest. Numerous techniques are available for amplifying nucleic acids. These techniques include polymerase chain reaction (PCR), ligase chain reaction (LCR), self-sustained sequence replication (3SR), nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA), transcription mediated isothermal CR cycling probe technology, and cascade rolling circle amplification (CRCA).
The analysis of nucleic acid targets conventionally comprises three steps: (1) the extraction/purification of the nucleic acid of interest from the biological specimen; (2) direct probe hybridization and/or amplification of the specific target sequence; and (3) specific detection thereof. In most conventional protocols these steps are performed separately, causing nucleic acid analysis to be labor intensive with each step requiring numerous manipulations, instruments, and reagents.
Qiagen, one market leader in nucleic acid sample preparation, produces and markets a variety of DNA and RNA sample preparation devices. Typically such devices are based upon glass fiber sheets where the biological sample must be clarified prior to its being applied to the binding matrix. The nucleic acid is typically captured in the presence of high salt buffer (anion exchange), the nucleic acid extensively washed, and the nucleic acid recovered by exposing the bound nucleic acid to a low ionic strength solution (e.g., Tris-EDTA (10 mM Tris-HCl, pH 7.5-8.0; 1 mM EDTA) or deionized water). The nucleic acid is then transferred to another vessel for amplification or further analysis. Other companies selling nucleic acid sample preparation devices include: Millipore (a membrane-based size exclusion ultra-filtration system), Promega, Bio-Rad, Invitrogen, and MWG (anion exchange-based systems).
Techniques for both purifying and amplifying nucleic acids on solid phase materials are known.
Solid-phase reversible immobilization (SPRI) is a widely used technique for purifying nucleic acids of interest. SPRI uses carboxyl-coated magnetic particles (that form the base material for most magnetic particle manufacture) to bind nucleic acids. Under conditions of high polyethylene glycol and salt concentration, SPRI magnetic particles have been found to bind both single- and double-stranded DNA, including PCR products. The nucleic acid typically may be eluted with water, 10 mM Tris or formamide.
Other types of functionalized particles may be used for binding template nucleic acid molecules, such as hydroxylated beads and reverse phase resins. These particles are available from a wide variety of commercial sources (e.g., Ansys, Waters, and Varian).
U.S. Pat. No. 4,921,805 discloses a capture reagent bound to a solid support useful for the separation and isolation of nucleic acids from complex unpurified biological solutions. The nucleic acid capture reagent comprises a molecule capable of intercalation into a DNA helix, and is attached to the solid support via a molecular linker. The capture reagent-nucleic acid complexes are isolated from the sample by centrifugation, filtration or by magnetic separation. Nucleic acids are separated from the isolated complexes by, for example, treating the capture reagent-nucleic acid complexes with dilute alkali.
Solid-phase amplification systems are also known.
The so-called DIAPOPS (Detection of Immobilized Amplified Product in One Phase System) combines solid phase PCR and detection by hybridization. DIAPOPS is used to covalently bind a PCR primer to a well. Nucleic acids are covalently bound to the solid phase by a carbodiimide condensation reaction. Manipulation is simplified and contamination diminished since the transfer of the amplicon from the amplification system to the detection system is eliminated.
‘Standard’ solid phase anchored amplification uses specific oligonucleotides coupled to a solid phase as primers for cDNA synthesis (prepared from a mRNA molecule). This amplification results in the production of a cDNA that is covalently linked to a solid phase such as agarose, acrylamide, magnetic, or latex beads. A solid phase with cDNA attached, generated using oligo (dT) as a primer, contains sequence information similar to a cDNA library; thus it represents a ‘solid phase library.’ The cDNA that is attached to the solid phase can be used directly as a template for PCR or can be modified enzymatically prior to the PCR or isothermic amplification procedure. Oligonucleotides that are attached to a solid phase can also serve for affinity purification of RNA. RNA isolated this way can be directly reverse transcribed, using the primer that is coupled to the solid phase. Subsequent amplification can employ this primer with or without additional internal primers. Since the cDNA is coupled to a solid phase, changing buffer conditions or primer composition is conveniently achieved by washing the solid phase and re-suspending in a different PCR mixture.
A simplified combined purification and amplification system is available from CpG-Biotech. This system utilizes a proprietary cell lysis solution (Release-IT™), which permits cell lysis and amplification to occur in the same reaction tube. Release-IT sequesters cell lysis products that might inhibit polymerases and improves the specificity and amplification yield. The CpG-Biotech Release-IT system eliminates the need for a separate genomic DNA purification step prior to amplification. The CpG-Biotech system makes use of a homogenous procedure.
Combined purification, amplification, and detection systems are also known in the art. Such systems permit isolation and purification of nucleic acids from complex samples, amplification of desired nucleic acids, and detection of the amplified products to all occur in a self-contained environment.
U.S. Pat. No. 5,955,351 discloses a self-contained device integrating nucleic acid extraction, amplification, and detection. The system integrates the extraction and amplification of the nucleic acids allowing both procedures to be performed in one chamber, detection in another chamber and collection of waste in yet another chamber. The reaction chambers are functionally distinct, sequential and compact. Xtrana, Inc. (Denver, Colo.) sells a commercial embodiment of such device, known as the SCIP cartridge. U.S. Pat. No. 6,153,425 similarly discloses a self-contained device integrating nucleic acid extraction, amplification and detection. Such device comprises a first hollow elongated cylinder with a single closed end and a plurality of chambers therein, and a second hollow elongated cylinder positioned contiguously inside the first cylinder capable of relative rotation. Sample is introduced into the second cylinder for extraction. The extracted nucleic acid is bound to a solid phase, and therefore not eluted from the solid phase by the addition of wash buffer. Amplification and labeling takes place in the second cylinder. Finally, the labeled, amplified product is reacted with microparticles conjugated with receptor specific ligands for detection of the target sequence.
A commercial product known as Xtra Amp™ (Xtrana, Inc., Denver, Colo.) permits nucleic acid extraction, amplification and detection to be performed in a single microcentrifuge tube. Xtra Amp employs a proprietary material, known commercially as Xtra Bind™, to extract and irreversibly bind nucleic acid in a sample. Xtra Bind binds both DNA and RNA in single strand form. Captured nucleic acid can be amplified directly on the solid phase by a variety of amplification strategies including those requiring single-strand initiation. Specific selection of low copy nucleic acid targets present in complex specimens can be performed by binding specific hybridization probes to the solid phase beads.
Carboxylated latex beads having a plurality of first and second nucleic acids are used in the so-called “Bridge Amplification” technique to similarly allow amplification, separation and detection in the same system. Such system is described in detail in U.S. Pat. No. 5,641,658, the disclosure of which is hereby incorporated by reference.
Other materials are also known to bind nucleic acids, albeit with less specificity. For example, nitrocellulose and polyamide membranes are often used as solid-phase nucleic acid transfer and hybridization matrices.
Presently, extensive use is made of polyamide matrices, in particular nylon matrices, as solid support for immobilization and hybridization of nucleic acids. Various types of polyamide matrices are known to bind nucleic acids irreversibly and are far more durable than nitrocellulose. As nucleic acids can be immobilized on polyamide matrices in buffers of low ionic strength, transfer of nucleic acids from gels to such matrices can be carried out electrophoretically, which may be performed if transfer of DNA by capillary action or vacuum is inefficient.
Two basic types of polyamide membranes are commercially available, unmodified nylon and charge-modified nylon. Charge-modified nylon is preferred for transfer and hybridization as its increased positively charged surface has a greater capacity for binding nucleic acids (See, e.g., U.S. Pat. No. 4,473,474, the disclosure of which is herein incorporated by reference). Nylon membranes must be treated, however, to immobilize the DNA after it has been transferred, as by way of thorough drying, or exposure to low amounts of ultraviolet irradiation (254 nm) and such immobilization is not irreversible.
Polyamide membranes, and in particular nylon membranes, offer many advantages in the filtration of materials in general. Nylon, as other polyamides, has a natural hydrophilicity, but a narrow wicking rate. It is also particularly strong. In particular, nylon can be cast as a liquid film and then converted to a solid film that presents a microporous structure when dried (See, e.g., U.S. Pat. No. 2,783,894). Such microporous structures permit micron and submicron size particles to be separated from fluid and provide an exceedingly high effective surface area for filtration. Microporous polyamide structures may be manufactured so as to be multizoned so as to provide for different filter characteristics in each zone (See, e.g., U.S. Pat. No. 6,090,441).
As taught in PCT/US98/07707, solid phase materials consisting of atoms or compounds of aluminum, as well as silicon and boron, when rendered sufficiently hydrophilic, such as by hydroxylation, irreversibly bind DNA and RNA, but not proteins. Such irreversible binding may be used to archive nucleic acids.